CHAPTER 2 Non-Volatile Floral Oils of Diascia spp. (Scrophulariaceae)*

Transcription

1 CHAPTER 2 (Scrophulariaceae)* Summary The floral oils of Diascia purpurea, D. vigilis, D. cordata, D. megathura, and D. integerrima (Scrophulariaceae) have been selectively collected from trichome elaiophores. The trimethylsilyl (TMS) derivatized floral oils were analyzed by electron impact (EI) gas chromatography-mass spectrometry (GC-MS), whilst the underivatized floral oil samples by electrospray Fourier-transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). The predominant compounds of floral oils from five Diascia spp. investigated are partially acetylated acylglycerols of (3R)-acetoxy fatty acids (C 14, C 16, and C 18 ), as was proven with synthetic reference sample. The mass spectral interpretation of significant compounds is presented in detail. The importance of Diascia floral oils for Rediviva bees is also discussed in a co-evolutionary context. *Based on a publication manuscript: Non-volatiles floral oils of Diascia spp. (Scrophulariaceae) (article in press) by authors: Kanchana Dumri, Lars Seipold, Jürgen Schmidt, Günter Gerlach, Stefan Dötterl, Allan G. Ellis and Ludger A. Wessjohann, Phytochemistry (doi: /j.phytochem ) 21

2 Results and Discussion 2.1. Fatty acid methyl ester (FAME) Profiling of the Diascia spp. Diascia oils from trichome elaiophores (Figure 2.1) are naturally yellowish. (A) (B) 1mm Figure 2.1. Diascia megathura (Scrophulariaceae): (A) inflorescence showing spurs (B) spur longitudinally split, showing the elaiophores with free oil. Arrows are showing the spurs (photos by G. Gerlach). Figure 2.2 illustrates the total ion chromatogram (TIC) of the FAME profiling of D. vigilis. The FAME profiling results of D. vigilis are presented in Table 2.1. Fatty acids and (3R)-hydroxy fatty acids with even-numbered chain length ranging from C 14 to C 18 represent the main compounds of the lipid collection. In all cases, there were no traces of acylglycerols due to the complete trans-esterification reaction. The main compound of all derivatized Diascia oil samples was (3R)-hydroxypalmitic acid (10, ca %) (Table 2.1). The EI mass spectra of TMS derivatives of 3-hydroxy fatty acid methyl ester show a poor molecular ion peak, but the molecular weight can be ascertained from a characteristic ion at m/z [M Me] +. Its formed by elimination of a methyl radical from the TMS group. The characteristic ions of oxygenated fatty acid TMS derivatives have common ions at m/z 73 ([SiMe 3 ] + ) and 89 ([SiMe 3 ] + ) (Curstedt 1974). A predominant ion at m/z 175, [Me(C)CH 2 CH(SiMe 3 )] + can be attributed to a cleavage between C 3 and C 4 of the carbon chain which is diagnostic of the hydroxyl group position of fatty acid chain (Mayberry 1980; Mielniczuk et al., 1992, 1993). 22

3 Non-Volatiles Floral ils of Diascia spp Relative Abundance (%) Retention Time (min) Figure 2.2. Total ion chromatogram (TIC) of the FAME profiling of D. vigilis floral oil (for the identification of compound members see Table 2.1, conditions GC1) 3-hydroxy fatty acids possess a chiral carbon. Due to the small amounts of samples available, only a chromatographic method is suitable to determine the absolute configuration. Thus diastereomeric derivatives were generated by esterification with optically pure (2S)-phenyl propionic acid from the acid chloride. The results were based on the GC-retention time comparison with a (2S)-phenylpropionyl derivative of a synthetic (see Appendix 1) (Hammarström 1975; Gradowska 1994; Weil et al., 2002; Seipold 2004). In most cases, the hydroxyl group at C-3 has (R)-configuration. The results were related to the fact of (R)-hydroxy family are intermediate during the fatty acid biosynthesis (Mayberry 1980). 23

4 Table 2.1. FAME profiling of Diascia spp. (as TMS derivatives a ). Relative composition (%) No. Compound b t R (min) D. purpurea D. vigilis D. cordata D. megathura D. integerrima 1 myristic acid palmitic acid stearic acid (3R)-hydroxymyristic acid (3R)-hydroxypalmitic acid (3R)-hydroxystearic acid a The examplary data were obtained from D. vigilis. b see Appendix 2 for EI-mass spectral data (Table A 2.1 and A 2.2). 24

5 2.2 GC/EI-MS analysis of the acylglycerols of Diascia oils A GC/EI-MS study of TMS derivatives of Diascia oils yielded both monoacylglycerols (MAGs) and diacylglycerols (DAGs) as main constituents along with small amounts of triacylglycerols (TAGs) (Table 2.2). According to the results obtained from the TMS derivatives, the detected acylglycerols of Diascia spp. contain one or two acetyl groups and (3R)-acetoxy fatty acid attached to the glycerol backbone. Furthermore, the ESI- FTICR-MS profiling analysis of underivatized Diascia oils confirmed the (3R)-acetoxy fatty acids, as long-chain moieties of the acylglycerols (see 2.3). The acetylation of the 3-hydroxy acids may be related to the export of the floral oils out of the cells. It has been reported that a hydroxyl group in fatty acids reduces the lipid transporter affinity compared to unfunctionalized fatty acids (Zachowski et al., 1998). Therefore, the acetylation could be crucial for an improved transport property (Seipold et al., 2004). Figure 2.3 shows the total ion chromatogram of the TMS derivative of D. vigilis floral oil. The identified compounds and relative composition of acylglycerols of the Diascia flower oils are summarized in Table 2.2. The key ions of the EI mass spectral data of the identified compounds are presented in Appendix 2. The two main components of D. vigilis floral oil are 2-[(3R)-acetoxypalmitoyl]glycerol (27) and 2-[(3R)- acetoxypalmitoyl]-1-acetylglycerol (30, Table 2.2) Relative Abundance (%) Retention Time (min) Figure 2.3. Total ion chromatogram (TIC) of TMS derivatives of D. vigilis floral oil (for the identification of compound members see Table 2.2, conditions GC1). 25

6 Table 2.2. Acylglycerols of the Diascia spp. identified as TMS derivatives by GC/EI-MS. Relative composition (%) No. t R (min) a Compound b D. purpurea D. vigilis D. cordata D. megathura D. integerrima [(3R)-acetoxymyristoyl]glycerol [(3R)-acetoxymyristoyl]glycerol [(3R)-acetoxymyristoyl]-1-acetylglycerol [(3R)-acetoxymyristoyl]-3-acetylglycerol ](3R)-acetoxymyristoyl]-1,3-diacetylglycerol unknown unknown [(3R)-acetoxypalmitoyl]glycerol [(3R)-acetoxypalmitoyl]glycerol [(3R)-acetoxypalmitoyl]-1-acetylglycerol [(3R)-acetoxypalmitoyl]-3-acetylglycerol [(3R)-acetoxypalmitoyl]-1,3-diacetylglycerol unknown [(3R)-acetoxystearoyl]glycerol [(3R)-acetoxystearoyl]glycerol [(3R)-acetoxystearoyl]-1-acetylglycerol [(3R)-acetoxystearoyl]-3-acetylglycerol [(3R)-acetoxystearoyl]-1,3-diacetylglycerol a obtained from D. vigilis, b see Appendix 2 for EI-mass spectral data (Table A 2.5, A 2.6, A 2.7 and A 2.15), (-) = not detected. 26

7 ur results indicated that the 1-monoacyl and 2-monoacyl isomer of (3R)-acetoxy fatty acids can be distinguished by their EI-MS data of the TMS derivatives. Figure 2.4 shows a comparison of the EI mass spectra of the 1-monoacyl and 2-monoacyl isomers of (3R)-acetoxypalmitoylglycerol. The mass spectra of the acylglycerol TMS derivatives show the characteristic ions at m/z 73, 89 and 103 corresponding to the fragments [SiMe 3 ] +, [SiMe 3 ] +, and [CH 2 SiMe 3 ] +, respectively (Curstedt 1974). Furthermore, the prominent ions at m/z 117 [CSiMe 3 ] + and 129 [CH 2 CHCHSiMe 3 ] + are commonly detected in the spectra of TMS derivatives of acylglycerols (Curstedt 1974; Wood 1980). The molecular weight of MAGs of (3R)- acetoxy fatty acids in Diascia spp. was deduced from the appearance of a significant ion type a, ([M Me HAc] +, Seipold 2004). In case of the MAGs of fatty acid, [M Me] + ion, formed by loss of methyl radical from the trimethylsilyl group represents the peak of highest mass (Curstedt 1974; Wood 1980). The ion at m/z 147 [Me 2 SiSiMe 3 ] + was due to the rearrangement ions, frequently detected in TMS derivative of monoacylglycerols. Scheme 2.1 shows the characteristic fragmentation of 2-[(3R)- acetoxypalmitoyl]glycerol (27) and 1-[(3R)-acetoxypalmitoyl]glycerol (28). An important key fragment of the 2-MAG isomer is the ion of type e at m/z 218 (20, 27, 35), while the ion of type (b HAc) is a typical fragment of 1-MAG (21, 28, 37). As previously suggested the 2-MAG displays a significant ion at m/z 218 which is formed by loss of the oxygenated fatty acid from the [M] + (Johnson and Holman 1966). Rearrangement of a TMS group from the acylglycerol backbone to the carboxyl group of the fatty acids leads to a c-type ion at m/z 311 (after loss of a HAc unit). The ion of type (d HAc) corresponds to the acylium ion after loss of 3-acetoxy group. Ion at m/z 203 (e Me) appears in both 1- and 2-monoacyl isomers. The most significant evidence of the 1-MAGs of (3R)-acetoxy fatty acid is the formation of an ion at m/z 369 (b HAc) as an unique peak, including the f-type ion at m/z 205. In most of the 1-MAGs, the ion of type b ([M 103] + ) corresponding to the loss of CH 2 Si(CH 3 ) 3, represents the base peak (Figure 2.4, see Appendix 2: Table A 2.5) (Johnson and Holman 1966; Curstedt 1974; Myher et al., 1974; Wood 1980). 27

8 (A) Relative Abundance (%) Ac e Me Si C H 27 (d HAc) (e _ Me) c a (b _ HAc) (B) Ac (d _ HAc) C 13 H 27 SiMe (e _ Me) f a c b m/z Figure ev-ei mass spectra of the TMS derivatives of monoacylglycerols (MAGs): (A) 2- [(3R)-acetoxypalmitoyl]glycerol (27) and (B) 1-[(3R)-acetoxypalmitoyl]glycerol (28). The significant fragment ions are described in Scheme 2.1. M + (27), m/z 532 (n.d.) 2-MAG _ Me _ HAc Ac C 13 H 27 -Me Me 2 Si d, m/z 297 (n.d.) e, [M _ R'CH], m/z 218 (e _ Me), m/z 203 _ HAc C 13 H [M _ Me _ HAc] + 27 Me 2 Si C 13 H 27 C 13 H 27 a, m/z 457 c, m/z 311 (d _ HAc), m/z 237 (b _ HAc), m/z 369 _ HAc -HAc _ HAc _ Me Ac C 13 H 27 SiMe 3 SiMe 3 Ac C 13 H 27 d, m/z 297 (n.d.) f, m/z 205 b, m/z 429 M + (28), m/z 532 (n.d.) 1-MAG Scheme 2.1. Mass spectral fragmentation of the monoacylglycerols 2-[(3R)- acetoxypalmitoyl]glycerol (27) and 1-[(3R)-acetoxypalmitoyl]glycerol (28) (n.d. = not detected). 28

9 Figure 2.5 illustrates the EI mass spectra of the TMS derivatives of 2-[(3R)- acetoxypalmitoyl]-1-acetylglycerol (30) and 1-[(3R)-acetoxypalmitoyl]-3-acetylglycerol (31). Generally, the mass spectral behavior of DAGs containing (3R)-acetoxy fatty acids and an acetyl moiety is similar to that of the MAGs. An ion at m/z 189 (g) appearing both in 1,2- and 1,3-DAGs can be explained as a cyclic structure (Curstedt 1974) as shown in Scheme 2.2. The TMS derivatives of 1,2-diacylglycerols (22, 30, 39) showed an analogous fragment at m/z 188 (type e) with moderate intensity (see Appendix 2: Table A 2.6). n the other hand, the e 1 -type ion (m/z 188, e 1 ) in 1,3-DAGs was of low abundance. This ion can be a first hint that the (3R)-acetoxy fatty acid was attached to the secondary hydroxyl group of the glycerol backbone. The ion k at m/z 145 confirming a 1,2-DAG was not observed in the TMS derivatives of 1,3-diacylglycerols (23, 31, 43) (Curstedt 1974). It should be pointed out that the ions at m/z 175 (type h) and m/z 146 (e 1 CH 2 C) only appear in the mass spectra of 1,3-diacylglycerols (Scheme 2.2, Table 2.2) (Seipold 2004). Relative Abundance (%) (A) Ac (d _ HAc) Ac k C g 13 H Me Si 55.2 c e a d h (B) 43.1 Ac C 13 H Ac 50 g (d _ HAc) (b _ HAc) (e _ 1 CH 2 C) a e 1 c m/z Figure ev-mass spectra of TMS derivatives of diacylglycerols (DAGs): (A) 2- [(3R)-acetoxypalmitoyl]-1-acetylglycerol (30) and (B) 1-[(3R)-acetoxypalmitoyl]-3- acetylglycerol (31). The significant fragment ions are described in Scheme 2.1 and

10 M + (30), m/z 502 (n.d.) 1,2-DAG _ Me _ HAc Ac e, [M _ R'CH] +,m/z 188 SiMe 3 k, m/z 145 [M _ Me _ HAc] + a, m/z 427 SiMe 3 g, m/z 189 H (e 1 _ CH 2 C), m/z 146 _ CH 2 C _ HAc _ Me Ac e 1, m/z 188 (n.s.) Ac h, m/z 175 M + (31), m/z 502 (n.d.) 1,3-DAG Scheme 2.2. Mass spectral fragmentation of the diacylglycerols 2-[(3R)- acetoxypalmitoyl]-1-acetylglycerol (30) and 1-[(3R)-acetoxypalmitoyl]-3-acetylglycerol (31) (n.d. = not detected; n.s. = not significant). The TAGs in floral oil of D. vigilis (24, 32, 44, Table 2.2) consist of one (3R)-acetoxy fatty acids (C 14, C 16 and C 18 ) at C-2 and two acetyl moieties at C-1 and C-3 of the glycerol backbone. Figure 2.6 illustrates the EI mass spectrum of 2-[(3R)- acetoxypalmitoyl]-1,3-diacetylglycerol (32). Mass spectra of these compounds show no molecular ion, but an ion at m/z [M 2HAc] + (a 1 ) as a peak of highest mass. Ion of type d was observed in very low abundance, whereas ion type (d HAc) was dominantly detected. The fragment at m/z 159 (e 2 ) was formed by loss of oxygenated fatty acid from the molecule which indicate the evidence of 1,3-diacetylglycerol (Vogel 1974). To confirm the diagnostic of fragments ion, the acetylation of [ 2 H]-labelled was 30

11 further performed (Scheme 2.3). The EI mass spectrum of [ 2 H]-labelled 2-[(3R)- acetoxypalmitoyl]-1,3-diacetylglycerol (32) is figured in Appendix 3 (Figure A 3.1). EI mass spectrum of [ 2 H]-labelled acetylated derivatives can certainly explain the potential sequence of the loss of acetoxy groups to form an ion at m/z [M 2HAc] + (a 1, m/z 355) which occur by [ 2 H 3 ] incorporation into the structure. Such an experiment gave evidence that the two acetoxy groups originate from the oxygenated fatty acid long chain and the second from glycerol backbone. The ion of type e 2 (m/z 159) is shifted by 6 mass units toward high masses in the [ 2 H 6 ]-labelled (2 CCD 3 ) derivative (Scheme 2.3, see also Figure A 3-1 in Appendix 3). This fragment confirmed a 1,3- diacetylglycerol which correspond to loss of the oxygenated fatty acid. EI mass spectra of 1-acyl-2,3-diacetyl-glycerols were also previously described (Reis et al., 2003) Ac Ac e Ac Ac Ac C 13 H 27 Relative Abundance (%) 50 0 d HAc d m/z a 1 Figure ev EI-mass spectrum of TMS derivative of triacylglycerol: 2-[(3R)- acetoxypalmitoyl]-1,3-diacetylglycerol (32). The significant fragment ions are described in Scheme 2.1 and

12 Ac Ac CH 3 C 13 H 27 D 3 CC D 3 CC CH 3 C 13 H 27 (32) [ 2 H]-labelled of 32 Scheme 2.3. [ 2 H]-acetylation of 2-[(3R)-acetoxypalmitoyl]-1,3-diacetylglycerol (32) (see EI-mass spectrum in Figure A 3.1, Appendix 3). The unidentified compounds 25, 26 and 33 are assumed to be isomers of 2-[(3R)- acetoxypalmitoyl]-1-acetylglycerol (30), 1-[(3R)-acetoxypalmitoyl]-3-acetylglycerol (31) and 1-[(3R)-acetoxystearoyl]-3-acetylglycerol (43), respectively (Table 2.2), but no unequivocal assignment is possible. An isomerization via an acyl-migration probably can occur during storage or measurement (Lyubachevskaya and Boyle-Roden 2000; Seipold 2004; Christie 2006). Therefore, the compounds 25, 26 and 33 might be also artefacts. In most cases, the floral oils of the five Diascia spp. exhibit a similar pattern with respect to their MAGs, DAGs and TAGs distribution, respectively. Exceptionally, in floral oil of D. cordata, TAGs could not be detected. Fatty acids were not detected by TMS derivatization of the floral oils of Diascia spp. DAGs represent the most abundant class (ca %) compared to MAGs (ca %) and TAGs (<15%) (Table 2.2). MAGs and DAGs as well as a small amount of TAGs were also described as the main oil components in Byrsonima crassifolia (Malpighiaceae) elaiophores (Vinson et al., 1997). The dominance of MAGs and DAGs is probably related to the insect digestive system. It has been shown that both MAGs and DAGs are better digestible than TAGs (Vinson et al., 1997). 32

13 2.3 Analysis of acylglycerols of underivatized Diascia oils The underivatized floral oils of the Diascia spp. are also investigated by ESI-FTICR- MS to obtain high resolution mass data of the lipid compounds. This will allow a rapid profiling of different oils in the future. All measurements were performed in the positive ion mode. In these cases, the electrospray mass spectra of the investigated oils show the sodium adducts ([M+Na] + ) of the corresponding compounds (Table 2.3). The positive ESI-FTICR mass spectrum of D. integerrima displays a DAG signal (base peak) that comprises the compounds 25, 26, 30 and 31 (m/z ) and contains even-number oxygenated fatty acid(s). The homology of investigated DAGs result in the compounds 22, 23 (m/z ) and compounds 33, 39, 43 (m/z ) (Figure 2.7). The MAGs signals represent the loss of an acetyl group (CH 2 C) from the DAGs, whereas TAGs show further an additional of acetyl moiety in their structures. Both of ESI- FTICR and GC/EI-MS results of Diascia floral oils represent the acetylated acylglycerols as the main compounds. In some case studied, such as ESI-FTICR-MS results of D. cordata floral oil noticeably indicated that TAGs consist long chain of (3R)-acetoxy fatty acids with chain lengths of C 14 and C 16 and two acetyl moieties, whereas the EI-MS data shows no hint of those TAGs. Likewise, fatty acids were not generally observed, when positive mode-esi was applied. However, fatty acids were easily detected and characterized by GC/EI-MS methods (see Table 2.1). The high resolution ESI-FTICR-MS results provide the high mass accuracy and elemental compositions of acetylated acylglycerols which usefully help the structural elucidation. However, the absolute abundance or even relative abundance of peaks in the ESI- FTICR does not certainly reflect the real proportions (Pulfer and Murphy 2003; Han and Gross 2005). Thus, in most cases, GC/EI-MS data were taken for an indication of the relative abundance of indicative compounds. Nonetheless, the most significant acylglycerols were detected in both methods. 33

14 a.i. 1.6e+07 25, 26, 30, e e e e+06 22, e+06 27, e+06 33, 39, e , 21 35, e m/z Figure 2.7. Positive-ion ESI-FTICR mass spectrum of the acylglycerol profile of D. integerrima. Peak heights are scaled relative to the highest magnitude peak (for the identified compounds see Table 2.3). 34

15 Table 2.3. Positive ion ESI-FTICR mass spectral data of the floral oils of the Diascia spp. a No. Compound type b Relative abundance (%) Elemental m/z Error MW Diascia Diascia Diascia Diascia Diascia composition ([M+Na] + ) (ppm) purpurea vigilis cordata megathura integerrima 20, 21 MAG (3-Ac 14:0) C 19 H 36 6 Na , 23 DAG (3-Ac 14:0, Ac) C 21 H 38 7 Na TAG (3-Ac 14:0, diac) C 23 H 40 8 Na , 26, 30, 31 DAG (3-Ac 16:0, Ac) C 23 H 42 7 Na , 28 MAG (3-Ac 16:0) C 21 H 40 6 Na TAG (3-Ac 16:0, diac) C 25 H 44 8 Na , 39, 43 DAG (3-Ac 18:0, Ac) C 25 H 46 7 Na , 37 MAG (3-Ac 18:0) C 23 H 44 6 Na TAG (3-Ac 18:0, diac) C 27 H 48 8 Na a The examplary data were obtained from D. integerrima (exception: compound 44 from D. purpurea ), b see full name of compounds in Table 2.2, MW= molecular weight, (-) = not detected. 35

16 In conclusion, positive ion ESI-FTICR mass spectra of the non-derivatized floral oils of Diascia spp. provided important results with respect to the acyglycerol profile. The determination of the elemental composition can provide a quick look at the lipid pattern of the oil-secreting Diascia flowers. With respect to the most prominent components were revealed in both the ESI-FTICR-MS and the GC/EI-MS experiments (Table 2.2 and 2.3). Based on these results, the related compositions of Diascia species indicate that they originate from the same evolutionary background as it is to be expected within a genus. It has been known that Diascia species are tightly associated with Rediviva oil collecting bees. The field observation revealed that variation in foreleg lengths of Rediviva bees can be explained as an evolutionary response to or rather a coevolutionary development with Diascia floral spur lengths (Steiner and Whitehead 1990, 1991). The floral oils play an important role in larval provision and have also been suggested to be used in nest construction (Cane et al., 1983; Buchmann 1987). Some hydroxylated fatty acids were reported to possess antibiotic properties (Valcavi et al., 1989; Weil et al., 2002). The prevalence of such chemical species in the Diascia flower oils instead of simple fatty acid oils may be necessary to keep the larval foods from microbial decomposition. The additional acetylation may either be required by the plant for excretion (Seipold et al., 2004) or reduced the water content of oil, or for the nest cell lining. Also, there is no detailed report on the chemistry of Rediviva bee nest cell lining. Therefore, some further investigations have to be carried out to verify the chemical nature of the association between Diascia flower oil and Rediviva bee cell lining. Further question concern the natural variation of flower oil compositions within a species or with flower age, the absolute configuration of acylglycerols with a chiral center at the sn-1 or sn-2 position, and if stereochemistry has any relevance in the biological context. 36